Blood First Edition Paper, prepublished online August 18, 2014; DOI 10.1182/blood-2014-04-571265
Full title: A nonsense mutation in IKBKB causes combined immunodeficiency Running title: IKBKB mutation causes Combined Immunodeficiency By: Talal Mousallem
1,2
2
3
*, Jialong Yang *, Thomas Urban , Hongxia Wang
2
2
2,5
3
Roberta E. Parrott , Joseph L. Roberts , David Goldstein , Rebecca H. Buckley Zhong
1
4
, Mehdi Adeli ,
2,5
, and Xiao-Ping
2,5
Department of Internal Medicine, Section of Pulmonary, Critical Care, Allergy and
Immunological Diseases, Winston-Salem, NC 27157, USA
2
Department of Pediatrics, Division of Allergy and Immunology, Duke University Medical Center,
Durham, NC 27710, USA
3
Center for Human Genome Variation, Duke University Medical Center, Durham, NC 27710,
USA
4
Hamad Medical Corporation, Doha, Qatar
4
Laboratory Medicine Center, Nanfang Hospital, Southern Medical University, Guangzhou,
Guangdong 510515, China
5
Department of Immunology, Duke University Medical Center, Durham, NC 27710, USA
*These authors contributed equally to this work.
Corresponding Author: Rebecca H. Buckley, M.D. Box 2898 or 362 Jones Building Duke University Medical Center Durham, North Carolina 27710 E-mail:
[email protected] Telephone #: 919/684-3204 Fax #: 919/681-7979
Copyright © 2014 American Society of Hematology
Key Points:
1.
A nonsense mutation in IKBKB caused the absence of IKK
β and lack of T and B cell
activation through their antigen receptors.
2.
β is not necessary for development of T or B lymphocytes but is important for their
IKK
activation and development/function of NK cells.
2
Abstract Identification of the molecular etiologies of primary immunodeficiencies has led to
important insights into the development and function of the immune system. We report here
the cause of Combined Immunodeficiency in 4 patients from 2 different consanguineous Qatari
families with similar clinical and immunologic phenotypes. The patients presented at an early
age with fungal, viral and bacterial infections and hypogammaglobulinemia. Although their B-
and T-cell numbers were normal, they had low regulatory T-cell and NK-cell numbers.
Moreover, patients’ T-cells were mostly CD45RA
+
naïve cells and defective in activation
following TCR stimulation. All patients contained the same homozygous nonsense mutation in
IKBKB (R286X) revealed by whole-exome sequencing with undetectable IKKβ and severely
decreased NEMO proteins. Mutant IKK
β(R286X) was unable to complex with IKKα/NEMO.
Immortalized patient B-cells displayed impaired I
κBα phosphorylation and NFκB nuclear
translocation. These data indicate that mutated IKBKB is the likely cause of immunodeficiency
in these four patients.
3
Introduction
Mutations in genes important in T-cell or in both T- and B-cell development and function
cause severe combined immunodeficiency (SCID), with a majority of cases caused by mutations
in IL2RG, IL7RA, ADA, JAK3, RAG1, RAG2, or DCLRE1C.
1;2
In contrast to SCID, T- and B-cell
development is not as severely impaired in combined immunodeficiency (CID). CID has been
associated with hypomorphic mutations in SCID-causing genes as well as mutations in several
other genes: ZAP70, MHC class II deficiencies , PNP, ORAI-1, STIM1, NEMO, CARD11 and
MALT1;
3-7
however, the etiology of many CID patients remains unknown.
The I
κB kinase (IKK) complex contains 2 structurally related catalytic subunits, IKKα and
IKKβ and a regulatory subunit, IKKγ/NEMO.
8;9
Null alleles of the X-linked gene encoding NEMO
(IKBKG) cause incontinentia pigmenti in heterozygous females and are lethal in hemizygous
males.
10
Hypomorphic alleles are compatible with life in males, but cause immunodeficiency
and accompanying developmental abnormalities of teeth, hair, or sweat glands in many
patients.
3;11;12
Hypermorphic mutations in the gene encoding IkB
α cause autosomal-dominant
ectodermal dysplasia with T-cell immunodeficiency, undetectable memory T-cells, and absence
of response to CD3-TCR activation.
13
The IKK
α/β/NEMO complex, activated by antigen and
other receptors, phosphorylates the IκB molecules, leading to subsequent NF
κB nuclear
translocation to activate transcription of genes involved in immune responses.
14
We report a
homozygous nonsense mutation in IKBKB in four CID patients from 2 unrelated families and
compare and contrast our findings with those in two other recent reports of mutations in this
gene.
15;16
4
Patient, Materials and Methods:
Abbreviated information is presented here. Details are provided in the Supplemental
Material.
Patients
All studies were performed with the approval of the DUMC Institutional Review Board
and written informed consent of the patients’ parents in accordance with the Declaration of
Helsinki. The patients were members of 2 unrelated consanguineous families. Select clinical
features are presented in the Table with further details in the Supplemental Materials.
Immunologic Phenotype Analysis
Flow cytometry of peripheral blood leucocytes was performed with labeled antibodies.
Lymphocyte proliferation was assessed as previously described.
17
Exome sequencing, alignment and variant calling
Exome sequencing was performed in the Genomic Analysis Facility in the Center for
Human Genome Variation (Duke University). Sequencing libraries were prepared from DNA
extracted from patients’ leukocytes using the Illumina TruSeq library preparation kit following
the manufacturer’s protocol.
Establishment of EBV cell lines
EBV-transformed B-cell lines were established from peripheral blood leukocytes as
previously described.
18
5
Cloning of full-length and mutant HuIKK β, transfection and retroviral transduction
β ) or R286X mutant IKKβ cDNA, amplified from a WT human IKKβ
WT full-length (FL-hIKK
template (Addgene) with a forward primer 5’-GTGAACCGTCAGAATTGATCT-3’ and a reverse
primer 5’-gagtGtttaaacACATCATGAGGCCTGCTCCA-3’ or 5’-
CggaattcTCAGGGGTGCCACATCAGCATCA-3’, was cloned into the MIGR1 retroviral vector.
Retroviral vectors were transfected into the Phoenix-Ampho cells to generate amphotropic
retroviruses.
Cell stimulation, isolation of nuclear and cytoplasmic fractions, and immunoblot analysis
EBV-transformed B-cells, rested in DPBS at 37°C for 30 minutes, were stimulated with PMA (20
ng/mL) or anti-human CD40 (10 μg/mL, Biolegend) at 37°C for 10 or 20 minutes. Nuclear
extracts, cytosolic or total cell lysates were subjected to standard immunoblotting analysis.
Real-time quantitative PCR
Total RNA from PBMCs and EBV-transformed B-cells were used for real-time
quantitative PCR analysis.
Statistical analysis
Two-tail Student’s t-test was performed (*P < .05; **P < .01; ***P < .001).
Results and discussion:
All 4 infants were hypogammaglobulinemic (Table 1). Three demonstrated normal or elevated
numbers of T-cells, normal numbers of B-cells, but low numbers of switched memory B-cells
6
+
and NK-cells. Most T-cells were CD45RA . There were abnormally low numbers of CD45RO
+
cells and CD4 CD25
bright
+
+
or CD4 FOXP3
+
T-
T-regulatory-cells (Treg) (Supplemental Figure 1).
All patient T-cells displayed normal responses to PHA, but low responses to PWM or Con
A in 3 of them (Figure 1A). Strikingly, all patients’ T-cells failed to respond to candida or tetanus
toxoid antigens or anti-CD3 stimulation (Figure 1B and 1C).
Moreover, patients’ CD4 T-cells
were impaired for TCR-induced CD25 and CD69 upregulation (Figure 1D).
NK-cell function,
tested in one patient from each family, was impaired in both (data not shown).
WES on patients 1 and 2 representing the 2 unrelated families identified a single
candidate nonsense homozygous variant in IKBKB (R286X). The other two patients and the
parents carried the same homozygous and heterozygous variant, respectively (Supplemental
Figure 1C and 1D), which was absent in the 998 sequenced controls and in the exome variant
server.
Contrarily to healthy controls and a heterozygous sibling, anti-N- or -C-terminal IKK
antibodies failed to detect any full-length nor truncated IKK
β
β
in patients’ PBMCs and EBV-
transformed B-cell lines (Figure 1E and 1F), which was not caused by possible low quality of the
antibody (Supplemental Figure 1E).
Interestingly, NEMO was virtually undetectable but IKK
was not obviously altered in patient samples (Figure 1E and 1F). Both I
levels were considerably increased in patients’ PBMCs (Figure 1G).
κKβ
α
and NEMO mRNA
Contrarily to WT IKK
β,
β(R286X) could not associate with either IKK α or NEMO when overexpressed in Pheonix-eco
IKK
cells (Figure 1H).
7
Patients’
EBV-transformed
B-cells
displayed
reduced
degradation, and impaired nuclear accumulation of NF
which activates the PKC-IKK-NF
reconstitution
defective I
1F).
than
with
κBα/NFκB
full-length
κB
pathway (Figure 1I).
but
not
β (R286X),
IKK
κB
19
I
κBα
phosphorylation
and
following stimulation with PMA,
Such defects were corrected after
suggesting
that
β (R286X)
IKK
caused
activation in patient-derived B-cells (Figure 1J and supplemental figure
Concordantly, expansion of patient’s EBV-transformed B-cells, which was slower in vitro
their
sibling’s
or
parents’
counterparts,
could
reconstituted with full-length but not mutant IKK
β(R286X)
expressing IKK
β(R286X)
IKK
β
be
accelerated
to
WT
levels
when
(Figure 1J). Interestingly, patient B-cells
expanded slower than cells expressing GFP alone, suggesting that
exerted a dominant-negative function. Currently, it is unclear if IKK
interfere with the residual IKK
β(R286X) may
α/NFκB signaling in these cells.
Together, these observations indicate that IKK
β protein is absent in the patients and that the C-
β mediates its association with IKKα/NEMO, which may be important for the
terminus of IKK
stability of itself and NEMO, supporting that the nonsense mutation in IKBKB caused CID in
these patients. Our results indicate that IKK
β is not required for the development of T- and B-
cells and show a novel cause of CID related to defects in the NF
κB pathway. These findings
highlight the diversity of phenotypes associated with mutations in different components of the
NF
κB pathway.
dysplasia,
20
For example, most patients with mutations in IKBKG had ectodermal
which was absent in our IKK
β(R286X) patients even though they have almost
undetectable NEMO. Interestingly, while this manuscript was in preparation/review, two other
IKBKB mutations in CID patients were reported, one duplication of exon 13 (IKBKB13Dup)
8
15
and
the other Y107X nonsense mutation of IKBKB.
15;16
The clinical features of our patients were
similar to those in the other two reports, with early infections with candida, gram negative
bacteria, viruses and mycobacteria. The immunological features were also similar. All have
elevated numbers of naïve T cells that were poorly activated by antigens and anti-CD3; low
numbers of B-cells that were also naïve, and low numbers and function of NK-cells. There was a
paucity of CD45RO positive T cells, Tregs and
γ/δ T-cells. Similar to our study, mutant IKKβ
protein was undetectable and NEMO was decreased in IKBKB13Dup patients.
was not obviously decreased in our IKK
IKBKB13Dup patients.
not reported.
15
15
However, IKK
α
β(R286X) patients but was severely decreased in the β(Y107X) mutation on IKKα and NEMO expression was
The effect of IKK
16
Acknowledgements.
The authors thank the Cancer Center flow-cytometry core facility at Duke
University for cell sorting. We would like to acknowledge the following individuals for the
contributions of control samples: Dr. Gianpiero Cavalleri, Dr. Norman Delanty, Dr. Chantal
Depondt, and Dr. Sanjay Sisodiya; Dr. William B. Gallentine, Dr. Erin L. Heinzen, Dr. Aatif M.
Husain, Ms. Kristen N Linney, Dr. Rodney A. Radtke, Dr. Saurabh R. Sinha, and Ms. Nicole M.
Walley; Dr. Julie Hoover-Fong, Dr. Nara L. Sobreira and Dr. David Valle; Dr. William L. Lowe; Dr.
Scott M. Palmer; Dr. Zvi Farfel, Dr. Doron Lancet, and Dr. Elon Pras; Mr. Arthur Holden and Dr.
Elijah Behr; Dr. Annapurna Poduri; Dr. Patricia Lugar; Dr. Rasheed Gbadegesin and Dr. Michelle
Winn; Dr. Robert Brown; Dr. Gianpiero Cavalleri, Dr. Norman Delanty, Dr. Chantal Depondt; Dr.
Yong-Hui Jiang, Dr. Vandana Shashi and Ms. Kelly Schoch; Dr. Eli J. Holtzman; Dr. Sarah Kerns
and Dr. Harriet Oster; Dr. Doug Marchuk; Dr. Demetre Daskalakis; Dr. Nicole Calakos; Dr. Francis
J. McMahon and Nirmala Akula; Dr. M Chiara Manzini.
9
Supported by the NHLBI GO Exome Sequencing Project (HL-102923, HL-102924, HL-102925, HL-
102926 and HL-103010) and by NIAID (AI076357, AI079088, and AI101206 to X-P.Z.) The
collection of control samples and the production of sequence data were funded in part by The
Epilepsy Phenome/Genome Project U01NS053998; Epi4K Project 1 – Epileptic Encephalopathies
U01NS077364; Epi4K Sequencing, Biostatistics and Bioinformatics Core U01NS077303; NIAID
Grant UO1AIO67854 (Center for HIV/AIDS Vaccine Immunology ("CHAVI")), and an award from
Biogen Idec.
Conflict of Interest Statements. None of the authors has any conflict of interest to declare. Authorship Contributions. TM designed the study, analyzed sequencing data, wrote the paper; JY designed and performed functional studies, contributed to manuscript preparation; TU
analyzed the sequencing data; HW participated in functional studies; MA referred the patients,
conducted immunologic investigations; REP performed extensive immunologic studies,
contributed to manuscript preparation; JLR contributed to data analysis and manuscript
composition; DG oversaw the whole genome sequencing and data analysis, contributed to
manuscript; RHB designed the study, wrote the paper, performed extensive immunologic
evaluations; XPZ designed functional studies, analyzed data, and wrote the paper.
10
Reference List
1. Fischer A, Le Deist F, Hacein-Bey-Abina S et al. Severe combined immunodeficiency. A model disease for molecular immunology and therapy. Immunol.Rev. 2005;203:98-109. 2. Buckley RH. Transplantation of hematopoietic stem cells in human severe combined immunodeficiency: longterm outcomes. Immunol.Res. 2011;49(1-3):25-43. 3. Orange JS, Brodeur SR, Jain A et al. Deficient natural killer cell cytotoxicity in patients with IKKgamma/NEMO mutations. J.Clin.Invest 2002;109(11):1501-1509. 4. Feske S, Gwack Y, Prakriya M et al. A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature. 2006;441(7090):179-185. 5. Picard C, McCarl CA, Papolos A et al. STIM1 mutation associated with a syndrome of immunodeficiency and autoimmunity. N.Engl.J.Med. 2009;360(19):1971-1980. 6. Jabara HH, Ohsumi T, Chou J et al. A homozygous mucosa-associated lymphoid tissue 1 (MALT1) mutation in a family with combined immunodeficiency. J.Allergy Clin.Immunol. 2013;132(1):151158. 7. Stepensky P, Keller B, Buchta M et al. Deficiency of caspase recruitment domain family, member 11 (CARD11), causes profound combined immunodeficiency in human subjects. J Allergy Clin Immunol 2013;131(2):477-485. 8. Zandi E, Rothwarf DM, Delhase M, Hayakawa M, Karin M. The IκB kinase complex (IKK) contains two kinase subunits, IKKα and IKKβ, necessary for IκB phosphorylation and NF-κB activation. Cell 1997;91(2):243-252. 11
9. Courtois G, Israel A. IKK regulation and human genetics. Curr.Top.Microbiol.Immunol. 2011;349:73-95. 10. Smahi A, Courtois G, Vabres P et al. Genomic rearrangement in NEMO impairs NF-κB activation and is a cause of incontinentia pigmenti. The International Incontinentia Pigmenti (IP) Consortium. Nature. 2000;405(6785):466-472. 11. Niehues T, Reichenbach J, Neubert J et al. Nuclear factor κB essential modulator-deficient child with immunodeficiency yet without anhidrotic ectodermal dysplasia. J Allergy Clin Immunol. 2004;114(6):1456-1462. 12. Orange JS, Levy O, Brodeur SR et al. Human nuclear factor κ B essential modulator mutation can result in immunodeficiency without ectodermal dysplasia. J.Allergy Clin.Immunol. 2004;114(3):650-656. 13. Courtois G, Smahi A, Reichenbach J et al. A hypermorphic IκBα mutation is associated with autosomal dominant anhidrotic ectodermal dysplasia and T cell immunodeficiency. J Clin Invest. 2003;112(7):1108-1115. 14. Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination: the control of NF-κB activity. Annu.Rev.Immunol. 2000;18:621-663. 15. Pannicke U, Baumann B, Fuchs S et al. Deficiency of innate and acquired immunity caused by an IKBKB mutation. N.Engl.J Med. 2013;369(26):2504-2514. 16. Burns SO, Plagnol V, Gutierrez BM et al. Immunodeficiency and disseminated mycobacterial infection associated with homozygous nonsense mutation of IKKβ. J Allergy Clin Immunol. 2014 Jul;134(1):215-218.e3. 12
17. Buckley RH, Schiff SE, Sampson HA et al. Development of immunity in human severe primary T cell deficiency following haploidentical bone marrow stem cell transplantation. J Immunol. 1986;136(7):2398-2407. 18. Sugden B, Mark W. Clonal transformation of adult human leukocytes by Epstein-Barr virus. J Virol. 1977;23(3):503-508. 19. Krishna S, Zhong X. Role of diacylglycerol kinases in T cell development and function. Crit Rev.Immunol. 2013;33(2):97-118. 20. Hanson EP, Monaco-Shawver L, Solt LA et al. Hypomorphic nuclear factor-κB essential modulator mutation database and reconstitution system identifies phenotypic and immunologic diversity. J.Allergy Clin.Immunol. 2008;122(6):1169-1177. 21. Shearer WT, Rosenblatt HM, Gelman RS et al. Lymphocyte subsets in healthy children from birth through 18 years of age: the Pediatric AIDS Clinical Trials Group P1009 study. J.Allergy Clin.Immunol. 2003;112(5):973-980.
13
Table 1: Clinical Features, Immunoglobulins and Lymphocyte Subsets Family 1 Patient 1
Patient 2
Family 2 Patient 3
Patient 4
Sex
Female
Male
Female
Male
Age at Presentation
5 mo.
11 mo.
7 mo.
6 mo.
Infections
Candida,
Candida,
Candida
BCG,
rotavirus,
Klebsiella,
BCG
CMV
Parameter
Normal Range
Clinical Features
Treatment
Candida,
Ablated, HLA-
Ablated,
Ablated, T
Ablated, T
identical
unrelated
cell depleted
cell
paternal
donor cord
paternal
depleted
marrow HSCT
blood HSCT
marrow
maternal
HSCT
marrow HSCT
Outcome
Alive and well
Died 2 mos.
Died 1 yr.
Died 13
later
later
mo. later
Immunoglobulins*
1
IgG
82
712
367
173
192-515
IgA
low
0
0
0
12-31
IgM
low
0
0
22
39-92
11,550(96)
14,792(96)
6,861(81)
9,118(82)
2,940-4,560
Flow Cytometry∞ T and NK Cells CD3
(49-76) CD4
8,656(72)
12,380(81)
5,946(70)
7,498(67)
1,860-3,360 (31-56)
CD8
2,881(24)
2,458(16)
1,262(15)
1,888(17)
11,118(93)
14,792(96)
6,852(81)
9,062(81)
α/β
11,502(96)
14,561(95)
6,683(79)
9,040(81)
P
3,000-4,680 (50-78)
Ti
P
720-1,440 (12-24)
CD28
P
Q
3,480-5,040 (58-84)
Q
γ/δ
180(2)
15(0)
42(1)
939(8)
0-540 (0-9)
CD16
120(1)
154(1)
644(8)
335(3)
180-1,080
Ti
CD4,CD25
bright
(3-18) 240(2)
ND
158(1.6)
ND
Q
24-336 (0.4-5.6)
Q
B Cells CD19
384(3)
461(3)
14
881(10)
894(8)
840-2,220
Q
(14-37) CD20
312(3)
476(3)
813(10)
950(9)
300-1,020 (5-17)
Switched Memory
2(13.3)
ND
2(9.2)
ND
B
P
Q
1-224 (22.4-79.4)
Q
Activation CD45
11,994(100)
15,345(100)
8,428(100)
10,526(94)
5,520-6,000 (92-100)
CD45RO,CD3
1063(9)
59(0)
521(8)
27(0)
177-1,140 (6-25)
CD45RA,CD3
9783(85)
14,274(97)
6,360(93)
8,589(94)
P
441-2,006 (15-44)
CD45RA,CD3,CD62L
8697(75.3)
ND
6,237(90.9)
ND
Q
Q
338-2,513 (11.5-55.1)
1 *Values are expressed as mg/dl. The patient was receiving IG replacement. ∞Values are expressed as cells/mm3 or (percentage of lymphocytes).
P
Normal values are the 95%
confidence intervals for ninety 6-12 mo-old healthy control subjects.
21 Q
Normal values are the
95% confidence intervals for 2338 healthy adult control subjects from the authors’ laboratory.
15
Q
Legend to Figure Figure 1. Contribution of IKKβ (R286X) mutation to CID. (A-C) Patients’ blood lymphocytes responded normally when stimulated with PHA but had variably low responses to Con A and
PWM (A). However, they failed to respond when stimulated with candida and tetanus antigens
(B) or soluble or immobilized anti-CD3 (C).
(D) Impaired upregulation of T cell activation
markers in patients’ CD4 T cells following overnight stimulation with plate-bound anti-CD3 or
PHA. (
E,F) Neither full-length nor truncated mutant IKKβ(R286X) protein is detectable in
patients (PT), siblings, and normal PBMC (E) and EBV-transformed B-cells (F) by immunoblotting
analysis with anti-N- and –C-terminal IKK
β antibodies. (G) mRNA levels of IKKα, IKKβ, and
NEMO in PMCs detected by real-time qPCR. (
IKK
α/NEMO.
H) IKKβ (R286X) is not able to form a complex with
Cell lysates from Phoenix-Eco cells transfected with either Flag-tagged-FL-IKK
β,
β(R286X), or vector control were subjected to immunoblotting analysis directly
Flag-tagged-IKK
(left panels) or after immunoprecipitation with anti-Flag antibody conjugated agarose beads
I
(right panels). ( ) Defective I
κBα/NFκB signaling in patient-derived B-cells can be reverted by
full-length (long) but not mutant IKK
β(R286X).
EBV-transformed B-cells of a sibling and patients
stably infected with retrovirus expressing WT IKK
β or with control vector were rested in PBS at
o
37 C for 30’ followed by PMA stimulation for 10 and 20 minutes. Immunoblotting analysis of
J
cytosolic fractions (top panel) or nuclear extracts (bottom panel) with indicated antibodies. ( )
Decreased expansion of patient-derived B-cells can be corrected by full-length but not mutant
β.
IKK
*P < .05; **P < .01; ***P < .001 determined by Student’s T-test.
16
Figure 1
15 10
8
C
Medium Candida Tetanus
6
15 CPM (x104)
20
4
Medium Anti-CD3 Immob anti-CD3
10
5
2 0 PT1
PT2
D
PT3
PT1
unsti
PT1
PT2
E WT
PT4
PT1
NC
PT3
% Max
80
60
60
40
40
20 0
35
20 0 102
103
104
105
0
CD69
0 102
103
104
105
IKK-β (N-ter)
CD25
IKK-β (C-ter)
H
G NC
IKK-β (C-ter) IKK-α NEMO β-actin
35 β-actin
PT+WT PT+vector sibling 0 10 20 0 10 20 0 10 20
*** flag
** 3.0
2.0
IKK-α
IKK-β
35
NEMO
IKK-α NEMO
J
sibling PT+WT PT+vector 0 10 20 0 10 20 0 10 20
Cell number (x104)
p-IκBα β-actin
100
35 IKK-α NEMO β-actin
***
IκBα
histone H3
flag
***
IKK-β
NF-κB p65
100
***
0.0
I
PT3
PT1
Sibling
6.0 Relative mRNA level
NC 100
sibling PT
NC
sibling PT
F IKK-β (N-ter)
NC
WT-IKKβ IKKβ(R286X) Vector
100
80
Vector
100
WT-IKKβ IKKβ(R286X)
PHA
PT4
IKK-α NEMO β-actin
100
PT3 anti-CD3
PT2
NC
0
NC
Sibling
PT4
PT3
PT3
PT1
PT2
NC
PT1
PT1 PT3 Sibling
0
NC
5
Sibling
CPM (x104)
B
Medium PHA Con A PWM
25
CPM (x104)
A
20
*** ***
15
**
10 5 0
PT + WT PT + mutant PT + vector
** ** * *
24h
48h
sibling PT
72h